Technical Field
The disclosure relates to a test slide. Particularly, the disclosure relates to a multiplex slide plate prefilled with polymerase chain reaction reagent(s).
Related Art
In the field of molecular biology, a variety of different experiments or test methods may be required to investigate the specific sample. For example, in the DNA assay, it is common to test a sample for several single-nucleotide polymorphism (SNP) genotypes, or the expression levels of a number of genes via polymerase chain reaction (PCR) assays. Several DNA assays may compose of a test panel for diagnosis, such as several PCR assays form a cancer diagnostic test panel. A PCR assay comprises at least two DNA specific primer probes (for some PCR also include additional target specific reporter probes), and this pair of primers has to correctly mix with the DNA template extracted from the sample to be tested (the sample) so as to examine the presence or the amount of the specific DNA targets in the sample. Traditionally, the pair of primers and the sample are delivered to the same reaction vessel for PCR. The delivery is usually done by pipetting the individual solutions from individual vials which store primer pair, enzyme and reagents, and the sample, to the reaction vessel. The most common vessel format is the 96-well titter plate. In such way, a PCR assay requires at least two pipettings, one for adding the primer pairs and another one for adding the sample to the reaction vessel. For example, for a panel to examine 36 targets in one sample, it needs at least 36 pipettings to add each pair of primers to 36 different reaction vessels, and another 36 pipettings to add sample to each of above vessels. This part of labour work is not only complex and error-prone, but also takes a lot of manpower.
One approach, called multiplexing approach, is to test the sample in the same reaction zone with mixtures of more than one pair of primers. Typically, 2 to 4 pairs of primers are added in one reaction vessel. For example, if 4 primer pairs using one vessel, the above mentioned example of test one sample for 36 targets, would require 9 reaction vessels, and the minimum pipetting needed would be 36 for adding primer pairs plus 9 for adding sample (a total of 45 pipettings). The labour work has been reduced. In spite of the convenience of the multiplexing approach, many reactions are performed simultaneously and the reaction and/or signal detection may interfere with each other to deteriorate the accuracy of the tests. Therefore, it is difficult to go beyond 6 pairs of primers in one reaction vessels.
Another approach is pre-filled primer pairs to individual reaction vessels in the factory. The lab user only needs to add sample to the pre-filled vessels. The above mentioned example of testing one sample for 36 targets would require only 36 pipettings for adding sample to 36 pre-filled vessels. Or even more the process may be further reduced to 9 pipettings, if multiplexing technique is also applied.
Another approach is to reduce the reaction vessel volume of titter plate to nano-liter range to save the cost of reagents. The result format is a slide-like micro-titter plate. The size and volume of reaction vessels (also called micro-wells or nano-wells), in a micro-titer plate are too small to be filled with the primers or sample manually without causing cross contamination between neighbouring vessels (i.e. the primer escapes from one well to other wells).
One approach is to provide the microfluidic chip designed with microfluidic channels, which deliver testing reagents (mainly primer pair) and samples to individual reaction wells for independent reactions. But the microfluidic chip is difficult to design and manufacture, and also adds high cost to the assays.
One approach is to pre-deliver primer pairs to each individual nano-wells in advance and immobilize the primers onto the well inter-surface. Therefore, the user can apply the sample to the wells by one pipetting or through one microfluidic channel without worrying about primers escaping from one well to other wells. The cross contamination between wells is minimized. However, the immobilization restricts the primer movement in the reaction vessel, which greatly reduce the efficiency of PCR.
In summary, if these primers probes may be placed into the reaction zone in advance, it can greatly simplify the labour work. However, as long as the reaction involving two or more reaction zones, the same sample has to be distributed to different zones of individual reactions without causing the pre-filled primers to cross contaminate between the zones.
The most important consideration is that each reaction vessel must be filled with the predetermined amount of sample. However, during filling the sample, the reaction vessels may not be cross-contaminated with reagents of different test assays. The traditional method is to use pipette or needle dispensers to load the sample “one by one” into the reaction wells. As the reaction well volume becomes smaller and the inter-well distance becomes closer, it is a challenge to fill the sample in each well without cross-contamination. Special mechanical mechanism or paths may be needed for the dispenser to reach each reaction well individually, which is complex and time consuming. As for the microfluidic devices designed with micro-channels, the design of the microfluidic devices and micro-pipeline in line significantly increases the production costs.